Ischemia-reperfusion injury (IRI) involving harvested organs and allograft transplantation may in part be caused by stimulation of a newly described innate pro-inflammatory immune system (i.e. cryopyrin-inflammasome, now referred to as NLRP-3 or NALP-3 inflammasome) that is known to cause secretion of pro-inflammatory cytokines (IL-1β) and induction of robust neutrophilic inflammation. Several biochemical triggers can stimulate the NALP-3 inflammasome and are referred to as danger associated molecular patterns (DAMPs), such as extracellular ATP, uric acid/urate (UA)/calcium pyrophosphate (CaPP) crystals, hyaluranon and intracellular hypokalemia. It is likely DAMPs are formed during IRI associated with transplantation pathobiology, as ischemia can induce anaerobic metabolism with metabolic acidosis from a combination of lactic acidosis, release of hydrogen ions during catabolism of ATP and accumulation of carbon dioxide. Reperfusion causes increased oxidants that can also lead to DAMPs formation and subsequent neutrophilic inflammation. Reduced pH disrupts mitochondrial ATP production, creating alterations in ion channels and membrane dissolution, resulting in intracellular sodium and calcium ion influx with potassium ion efflux and subsequent intracellular hypokalemia. Extracellular ATP can also stimulate purinergic P2X membrane receptors and cause further efflux of potassium ions from cells. Purine catabolism of nucleic acids and ATP in stressed and dying cells is associated with increased concentrations of UA that can crystallize at lower pH. ATP utilization during anaerobic metabolism leads to formation of pyrophosphates, which can combine with calcium ions to form CaPP. Moreover, released ATP from cytotoxic injury can bind to cryopyrin (i.e. putative protein of NALP-3 inflammasome) which has ATPase activity and can cause further increased production of UA and pyrophosphates. Consequently, these biochemical events circumstantially support the likelihood that IR transplantation pathobiology can induce DAMPs.
The NALP-3 inflammasome is located in the cytoplasm of various cell types (monocytes, neutrophils, mast cells, dendritic cells, macrophages, glia and chondrocytes) and following DAMPs stimulation, can activate caspase-1 to catalyze stored pro-IL-1β to IL-1β, with subsequent secretion of IL-1β. This pro-inflammatory cytokine in turn can provoke a cascade of pro-inflammatory events (i.e. up-regulation of vascular adhesion molecules (ICAM), IL-6 release, increased neutrophil and monocyte chemokines, IL-17 A secretion), that can induce marked neutrophilic inflammation in IRI. Since a central part of transplantation pathobiology is associated with IRI, it is posited that secretion of IL-1β by DAMPs stimulation of NALP-3 inflammasomes may play a significant role in causing neutrophilic inflammation and thereby reduce viability of harvested organs and increase allograft rejection.
Research involving rare autosomal dominant auto-inflammatory disorders led to the original discovery of the NALP-3 inflammasome. Hoffman et. al first identified the cryopyrin-encoding gene on chromosome 1q44 by investigating Familial Cold Auto-Inflammatory Syndrome (FCAS), a rare autosomal dominant syndrome characterized by cold exposure induction of IL-1β secretion that causes fever, neutrophilic leukocytosis, acute phase reactant elevations, and neutrophil leukocyte infiltrated dermatosis. The cryopyrin gene discovery led to recognition of the NALP-3 inflammasome by Martinon et al, a cytoplasmic macromolecular protein complex containing the putative protein cryopyrin and other adaptor proteins. Two other rare and seemingly unrelated autosomal dominant periodic fever syndromes (i.e. Muckle-Wells and Neonatal Onset Multisystem Inflammatory Disease, i.e. NOMID) with dysregulated elevated 1 L-1β production and profound neutrophilic inflammation were found to have mutations on the same gene. All three auto-inflammatory hereditary syndromes, referred to as cryopyrin-associated periodic syndromes (CAPS), are exceptionally responsive to IL-1β targeted therapy (IL-1β TT), which provided unequivocal evidence for IL-1β mediation of robust neutrophilic inflammation in human disorders. There is further evidence to suggest that blocking IL-1β can not only diminish neutrophilic inflammation in CAPS but also in several other IL-1β mediated neutrophilic inflammatory disorders, such as recalcitrant gout, Still's juvenile arthritis and Schnitzler's syndrome. More recently, autosomal recessive syndromes with deficiency of naturally occurring IL-1β receptor antagonists have been described. These syndromes exhibit manifestations of unopposed IL-1β mediated neutrophilia with pustulosis and osteomyelitis, which are also very responsive to IL-1β targeted therapy (IL-1β TT). The excellent response to IL-1βTT in the aforementioned conditions provides the rationale for its consideration in treating IRI transplantation pathobiology characterized by IL-1β secretion and dominant neutrophilic inflammation, as observed in many animal and human IR models following de-oxygenation stress.
It is known that IL-1β secretion can occur following pathogen associated molecular pattern (PAMPs) stimulation of innate immune receptors, such as cytoplasmic receptors (i.e. NALP-3 inflammasome, NALP-1 inflammasome, and IPAF inflammasome) and membrane associated receptors referred to as toll-like receptors (TLRs′). It is likely that PAMPs stimulation of innate immune receptors explain some IL-1β secretion and neutrophilic inflammation in infectious diseases, such as post-operative infectious disease complications following organ transplantations. However in contrast, many healthy animal models of IRI (i.e. cerebral, renal, intestinal and an ex-vivo human cardiac model), with no evidence of detectable PAMPs have demonstrated secretion of IL-1β within one hour of de-oxygenation after arterial blood vessel occlusion or during ex-vivo cardiac tissue perfusion with buffered fluids. Subsequent development of prominent neutrophilic inflammation was consistently observed in the arterial blood vessel occlusion studies and in several of these models, IL-1β targeted therapy (IL-1β TT) reduced the extent of the neutrophilic inflammation and injury. Until recently, there has been no reasonable explanation for induction of IL-1β secretion and neutrophilic inflammation following de-oxygenation in IR models, absent detectable PAMPs. Although PAMPs could have been present in undetectable concentrations in these animal experiments, it remains unclear why low concentrations of PAMPs would stimulate IL-1β secretion and cause neutrophilic inflammation only after arterial occlusion. Giamarellos-Bourboulis et al provided an interesting observation as monocytes challenged by urates in combination with LPS caused synergistic secretion of IL-1β, suggesting that even low and possibly undetectable concentrations of PAMPs may require DAMPs to induce IL-1β secretion in certain circumstances, such as following arterial occlusion.